Improved antisense oligonucleotide induced exon skipping in the mdx mouse model of muscular dystrophy
Identifieur interne : 000D51 ( Istex/Checkpoint ); précédent : 000D50; suivant : 000D52Improved antisense oligonucleotide induced exon skipping in the mdx mouse model of muscular dystrophy
Auteurs : Christopher J. Mann ; Kaite Honeyman ; Graham Mcclorey ; Sue Fletcher ; Stephen D. Wilton [Australie]Source :
- The Journal of Gene Medicine [ 1099-498X ] ; 2002-11.
English descriptors
- Teeft :
- Acad, Antisense, Antisense oligonucleotides, Assay, Cells transfected, Consistent exon, Copyright, Differentiation conditions, Digestion, Donor splice site, Dos, Duchenne, Dystrophin, Dystrophin exon, Dystrophin expression, Dystrophin gene, Dystrophin levels, Dystrophin mrna, Dystrophin protein, Dystrophin synthesis, Dystrophy, Exon, Gene, Gene therapy, Genet, Heteroduplexes, Individual dishes, Intron, Intron junction, John wiley sons, Lipofectin, Mouse model, Mrna, Mrna transcripts, Muscle cells, Muscular dystrophy, Mutation, Natl, Nonsense mutation, Normal cells, Nuclease, Nuclease digestion, Nucleic acids, Oligonucleotides, Plating, Primer, Proc, Proc natl acad, Protein levels, Single dose, Single transfection, Splice, Splice site, Splice sites, Target site, Transcript, Transfected, Transfection, Untreated, Vast improvements, Western australia.
Abstract
Background: Duchenne muscular dystrophy (DMD) is a fatal genetic disorder caused by dystrophin gene mutations that preclude synthesis of a functional protein. One potential treatment of the disorder has utilised antisense oligoribonucleotides (AOs) to induce removal of disease‐associated exons during pre‐mRNA processing. Induced in‐frame mRNA transcripts encode a shorter but functional dystrophin. We have investigated and improved the design of AOs capable of removing exon 23, and thus the disease‐causing nonsense mutation, from mRNA in the mdx mouse model of DMD. Methods: H−2Kb‐tsA58 mdx cultures were transfected with complexes of Lipofectin and AOs. Exon skipping was detected by RT‐PCR and subsequent protein production was demonstrated by Western blotting. AOs were delivered at a range of doses in order to compare relative efficiencies. Results: We describe effective and reproducible exon 23 skipping with several AOs, including one as small as 17 nucleotides. Furthermore, the location of a sensitive exon 23 target site has been refined, whilst minimum effective doses have been estimated in vitro. These doses are significantly lower than previously reported and were associated with the synthesis of dystrophin protein in vitro. Conclusions: These results demonstrate the increasing feasibility of an AO‐based therapy for treatment of DMD. By refining AO design we have been able to reduce the size and the effective dose of the AOs and have dramatically improved the efficiency of the technique. Copyright © 2002 John Wiley & Sons, Ltd.
Url:
DOI: 10.1002/jgm.295
Affiliations:
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Wilton</name><affiliation></affiliation><affiliation wicri:level="1"><country wicri:rule="url">Australie</country></affiliation><affiliation></affiliation></author></analytic><monogr></monogr><series><title level="j" type="main">The Journal of Gene Medicine</title><title level="j" type="alt">JOURNAL OF GENE MEDICINE, THE</title><idno type="ISSN">1099-498X</idno><idno type="eISSN">1521-2254</idno><imprint><biblScope unit="vol">4</biblScope><biblScope unit="issue">6</biblScope><biblScope unit="page" from="644">644</biblScope><biblScope unit="page" to="654">654</biblScope><biblScope unit="page-count">11</biblScope><publisher>John Wiley & Sons, Ltd.</publisher><pubPlace>Chichester, UK</pubPlace><date type="published" when="2002-11">2002-11</date></imprint><idno type="ISSN">1099-498X</idno></series></biblStruct></sourceDesc><seriesStmt><idno type="ISSN">1099-498X</idno></seriesStmt></fileDesc><profileDesc><textClass><keywords scheme="Teeft" xml:lang="en"><term>Acad</term><term>Antisense</term><term>Antisense oligonucleotides</term><term>Assay</term><term>Cells transfected</term><term>Consistent exon</term><term>Copyright</term><term>Differentiation conditions</term><term>Digestion</term><term>Donor splice site</term><term>Dos</term><term>Duchenne</term><term>Dystrophin</term><term>Dystrophin exon</term><term>Dystrophin expression</term><term>Dystrophin gene</term><term>Dystrophin levels</term><term>Dystrophin mrna</term><term>Dystrophin protein</term><term>Dystrophin synthesis</term><term>Dystrophy</term><term>Exon</term><term>Gene</term><term>Gene therapy</term><term>Genet</term><term>Heteroduplexes</term><term>Individual dishes</term><term>Intron</term><term>Intron junction</term><term>John wiley sons</term><term>Lipofectin</term><term>Mouse model</term><term>Mrna</term><term>Mrna transcripts</term><term>Muscle cells</term><term>Muscular dystrophy</term><term>Mutation</term><term>Natl</term><term>Nonsense mutation</term><term>Normal cells</term><term>Nuclease</term><term>Nuclease digestion</term><term>Nucleic acids</term><term>Oligonucleotides</term><term>Plating</term><term>Primer</term><term>Proc</term><term>Proc natl acad</term><term>Protein levels</term><term>Single dose</term><term>Single transfection</term><term>Splice</term><term>Splice site</term><term>Splice sites</term><term>Target site</term><term>Transcript</term><term>Transfected</term><term>Transfection</term><term>Untreated</term><term>Vast improvements</term><term>Western australia</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">Background: Duchenne muscular dystrophy (DMD) is a fatal genetic disorder caused by dystrophin gene mutations that preclude synthesis of a functional protein. One potential treatment of the disorder has utilised antisense oligoribonucleotides (AOs) to induce removal of disease‐associated exons during pre‐mRNA processing. Induced in‐frame mRNA transcripts encode a shorter but functional dystrophin. We have investigated and improved the design of AOs capable of removing exon 23, and thus the disease‐causing nonsense mutation, from mRNA in the mdx mouse model of DMD. Methods: H−2Kb‐tsA58 mdx cultures were transfected with complexes of Lipofectin and AOs. Exon skipping was detected by RT‐PCR and subsequent protein production was demonstrated by Western blotting. AOs were delivered at a range of doses in order to compare relative efficiencies. Results: We describe effective and reproducible exon 23 skipping with several AOs, including one as small as 17 nucleotides. Furthermore, the location of a sensitive exon 23 target site has been refined, whilst minimum effective doses have been estimated in vitro. These doses are significantly lower than previously reported and were associated with the synthesis of dystrophin protein in vitro. Conclusions: These results demonstrate the increasing feasibility of an AO‐based therapy for treatment of DMD. By refining AO design we have been able to reduce the size and the effective dose of the AOs and have dramatically improved the efficiency of the technique. Copyright © 2002 John Wiley & Sons, Ltd.</div></front></TEI><affiliations><list><country><li>Australie</li></country></list><tree><noCountry><name sortKey="Fletcher, Sue" sort="Fletcher, Sue" uniqKey="Fletcher S" first="Sue" last="Fletcher">Sue Fletcher</name><name sortKey="Honeyman, Kaite" sort="Honeyman, Kaite" uniqKey="Honeyman K" first="Kaite" last="Honeyman">Kaite Honeyman</name><name sortKey="Mann, Christopher J" sort="Mann, Christopher J" uniqKey="Mann C" first="Christopher J." last="Mann">Christopher J. Mann</name><name sortKey="Mcclorey, Graham" sort="Mcclorey, Graham" uniqKey="Mcclorey G" first="Graham" last="Mcclorey">Graham Mcclorey</name></noCountry><country name="Australie"><noRegion><name sortKey="Wilton, Stephen D" sort="Wilton, Stephen D" uniqKey="Wilton S" first="Stephen D." last="Wilton">Stephen D. Wilton</name></noRegion></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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